1. Run your SDS-polyacrylamide Laemelli gel and blot the protein bands onto nitrocellulose membrane, following standard methods (see chapter 12 of Harlow and Lane, 1988). Be sure NOT to use PVDF or other nylon-based membranes, as chicken antibodies tend to bind non-specifically, contributing to higher backgrounds.

2. Block non-specific sites on your membrane by incubating the membrane for 30 min at room temperature with BlokHen® (diluted 1:10 with water) using gentle agitation.

3. Wash the blot three times in PBS with 0.05% Tween-20 at room temperature. Each wash should be at least 10 minutes with gentle agitation.

4. Incubate your blot for at least 1 hour at room temperature with your chicken antibody diluted in “antibody dilution buffer.” Optimal dilutions of chicken antibody may be as low as 1:500 or as high as 1:50,000 depending on the immunogenicity of the antigen. Generally, the longer the period of incubation, the better (provided that the blot doesn’t dry out) — even overnight incubations are fine.

8. Develop the blot for 5-20 minutes at room temperature using Western Blue Stabilized AP Substrate®.

* These methods have been adapted from a number of sources, including “Antibodies: A Laboratory Manual” by Harlow and Lane (Cold Spring Harbor Press, 1988), “Current Protocols in Molecular Biology” by Ausubel et al (Wiley & Sons, 1997), and various Specification Sheets offered by manufacturers. We highly recommend the manual by Harlow and Lane, which provides general information on antibodies. Other useful resources on antibodies can be found on our Links to Other Websites page.

1. Run your SDS-polyacrylamide Laemelli gel and blot the protein bands onto nitrocellulose membrane, following standard methods (see chapter 12 of Harlow and Lane, 1988). Be sure NOT to use PVDF or other nylon-based membranes, as chicken antibodies tend to bind non-specifically, contributing to higher backgrounds.

2. Block non-specific sites on your membrane by incubating the membrane for 30 min at room temperature with BlokHen® (diluted 1:10 with water) using gentle agitation.

3. Wash the blot three times in PBS with 0.05% Tween-20 at room temperature. Each wash should be at least 10 minutes with gentle agitation.

4. I ncubate your blot for at least 1 hour at room temperature with your chicken antibody diluted in “antibody dilution buffer.” Optimal dilutions of chicken antibody may be as low as 1:500 or as high as 1:50,000 depending on the immunogenicity of the antigen. Generally, the longer the period of incubation, the better (provided that the blot doesn’t dry out) — even overnight incubations are fine.

8. Develop the blot for 5-10 minutes at room temperature using SuperSignal® HRP Substrate Working Solution.

9. Place the blot against X-ray film and exposure from 10 seconds to 5 minutes (depending on the intensity of the signal).

10. Develop the X-ray film as you normally would.

NOTE: Chicken antibodies tend to display higher backgrounds on polyvinylidene fluoride (PVDF) and other nylon-based membranges. For this reason, we recommend using nitrocellulose membranes for western blotting applications.

* These methods have been adapted from a number of sources, including “Antibodies: A Laboratory Manual” by Harlow and Lane (Cold Spring Harbor Press, 1988), “Current Protocols in Molecular Biology” by Ausubel et al (Wiley & Sons, 1997), and various Specification Sheets offered by manufacturers. We highly recommend the manual by Harlow and Lane, which provides general information on antibodies. Other useful resources on antibodies can be found on our Links to Other Websites page.

Immunocytochemistry using Fluorescein-labeled Secondary Antibodies

fluorescein anti-fading reagent — Make up a 2 mg/ml phenylene diamine solution in PBS (phenylene diamine requires extensive vortexing to put it into solution). Once the phenylene diamine is completely dissolved, add an equal volume of glycerol and mix. This reagent will last about a week at -20C. Discard this reagent when it starts to turn dark brown.

4. Incubate your sections or cells with your chicken primary antibodies (diluted in “antibody dilution buffer”) for at least 1 hour at room temperature. The concentration of your antibody may be anywhere from 1:100 to 1:50000, depending on the titre of the antibody and the concentration of your antigen.

5. Wash your sections or cells over a 10 minute period at room temperature (with two changes of PBS).

6. Incubate your sections or cells with fluorescein-labeled goat anti-chicken IgY (1:500 dilution in “antibody dilution buffer) for 1 hour at room temperature. Be sure to keep these slides or culture dishes in subdued light (e.g., in a drawer) to avoid bleaching of the fluorescein dye.

7. Repeat step #5.

8. Add a drop of “fluorescence anti-fading reagent” to your sections or cells. Place a coverslip over the section. If you want to reduce messiness, you may also seal the coverslip by painting the edges with nail polish.

9. Store the slides or culture dishes in the refrigerator (in the dark).

* These methods have been adapted from a number of sources, including “Antibodies: A Laboratory Manual” by Harlow and Lane (Cold Spring Harbor Press, 1988), “Current Protocols in Molecular Biology” by Ausubel et al (Wiley & Sons, 1997), and various Specification Sheets offered by manufacturers. We highly recommend the manual by Harlow and Lane, which provides general information on antibodies. Other useful resources on antibodies can be found on our Links to Other Websites page.

Immunoprecipitation

(based on methods described in Current Protocols in Molecular Biology, F. Ausubel et al., John Wiley & Sons)

Note: Chicken IgY’s lack the Fc domain where proteins A and G normally bind. Consequently, protein A/G-Agarose or -Sepharose cannot be used to immunoprecipitate chicken IgY’s. There are, however, three alternative approaches to get around this limitation:

First, one can substitute PrecipHen for protein G-Agarose. This product is a goat anti-chicken IgY antibody conjugated to agarose beads. For more information about this product, click here.

Second, one can include an extra step between chicken IgY binding to antigen and addition of protein G-Sepharose. In this step, you simply incubate the chicken IgY/antigen complex with goat anti-chicken IgY. In this way, the protein G-Agarose immunoprecipitates the goat antibody bound to the chicken antibody bound to the antigen. For more information about our goat anti-chicken IgY, click here.

Third, one can conjugate your affinity-purified chicken antibodies directly to Agarose. This can be accomplished using CarboLink kit from Pierce (Catalog #44900). For more information about this product, click here to reach the Pierce on-line catalog.

1. Lyse your cells or tissue using Lysis Buffer (4C). Centrifuge the lysate at 3000 g for 15 minutes at 4C to pellet nuclei and other cellular debris.

2. Transfer the supernatant to a set of microfuge tubes and centrifuge at 10,000 g for 40 minutes at 4C to remove smaller membranous material.

3. Transfer the supernatant to another set of microfuge tubes and add a slurry of PrecipHen® to the tube. Typically, a volume of 200 ul (a packed bed volume of 100 ul plus 100 ul of PBS) is sufficient for this step. Incubate this mixture for 30 minutes at 4C with gentle agitation to keep the PrecipHen® in suspension. Finally, centrifuge the PrecipHen® to remove any proteins in your sample that happen to bind non-specifically to either the agarose or the goat antibody.

4. Transfer the supernantant to another set of microfuge tubes, and add your primary chicken antibody to the tube. Incubate 1-2 hours on ice. [NOTE: In most cases, the primary antibody needs to be affinity-purified. This is because only a small fraction (less than 1%) of the antibody in purified IgY fractions is likely to be against the antigen of interest. Consequently, only a small fraction of the antibodies in IgY fractions contribute to specific binding of the protein of interest, whereas the other antibodies contribute to non-specific binding. In contrast, 100% of affinity-purified antibodies recognize the protein of interest, greatly improving signal-to-noise ratios.]

5. Add a slurry of PrecipHen® to the tube. [NOTE: The amount of PrecipHen to be added depends on the total amount of chicken IgY present in the tube. Since the binding capacity of 1.0 millilter PrecipHen (packed volume) is 1 mg of chicken IgY, one should add approximately twice the binding capacity of the PrecipHen. In other words, if you added 50 micrograms of primary chicken IgY to each microfuge tube in step 4 above, you would want to add 100 microliters (packed volume) of Preciphen to that tube. Since PrecipHen is a 1:1 slurry in PBS, this means that you would add a 200 ul slurry volume to each tube.]

6. Incubate for 3 hours – overnight at 4C with gentle agitation.

7. Centifuge the slurry in a refrigerated microfuge for 5 minutes at 4C. Discard the supernatant. Resuspend the slurry in Washing Buffer A. Incubate for 5 minutes with gentle agitation.

8. Repeat step 7.

9. Centifuge the slurry in a refrigerated microfuge for 5 minutes at 4C. Discard the supernatant. Resuspend the slurry in Washing Buffer B. Incubate for 5 minutes with gentle agitation.

10. Repeat step 9.

11. Centifuge the slurry in a refrigerated microfuge for 5 minutes at 4C. Discard the supernatant. Resuspend the slurry in Washing Buffer C. Incubate for 5 minutes with gentle agitation.

12. Repeat step 11.

13. Centrifuge the slurry in a refrigerated microfuge at 4C. Discard the supernatant.

14. Add Laemmli buffer. Place the tube in boiling water for 5 minutes. Be sure to poke a small hole in the cap of the tube to prevent built-up of gases.

15. Vortex the microfuge tube briefly, and then centrifuge it again for 5 minutes at 4C in a microfuge.

16. Finally, load the supernatant onto an SDS-polyacrylamide gel, subject the gel to electrophoresis, and transfer the proteins onto a suitable membrane. This membrane is now ready for western blotting with a second antibody against the protein of interest.

Note: If you notice high backgrounds, substitute Lysis Buffer for Washing Buffer A in steps 7 and 9, above.

* These methods have been adapted from a number of sources, including “Antibodies: A Laboratory Manual” by Harlow and Lane (Cold Spring Harbor Press, 1988), “Current Protocols in Molecular Biology” by Ausubel et al (Wiley & Sons, 1997), and various Specification Sheets offered by manufacturers. We highly recommend the manual by Harlow and Lane, which provides general information on antibodies. Other useful resources on antibodies can be found on our Links to Other Websites page.

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